PROJECT SUMMARY/ABSTRACT Microvilli are actin-based protrusions located at the apical surface of transporting epithelial cells. In the context of the mammalian intestine, microvilli collectively comprise the intestinal brush border, which acts as a first line of defense against pathogens and increases the surface are for nutrient absorption. Despite the importance of microvilli for intestinal health and human viability, little is known about the molecular events that underlie microvillar assembly. Early ultrastructural analysis by transmission electron microscopy revealed that the distal tips of microvilli are occupied by an electron dense plaque known as the ?distal tip complex?, which embeds the barbed ends of actin filaments. As the barbed ends of actin filaments are the preferred site of actin monomer addition, proteins that regulate actin filament length typically target to these ends. One candidate distal tip complex protein is epidermal growth factor receptor pathway substrate 8 (EPS8). Intestinal tissue staining of EPS8 revealed striking distal tip localization along the length of the intestinal crypt-villus. Additionally, the domain architecture of EPS8 lends the potential to bind plasma membrane, actin, and other signaling factors, features that position it as an attractive candidate for orchestrating microvillar assembly. Moreover, studies have shown that loss of EPS8 in both cell culture and mouse knockout models results in shortened microvilli. Despite these findings, how EPS8 promotes microvillar growth remains unknown. Thus, this proposal seeks to define the mechanism by which EPS8 promotes microvillar growth using live cell, super resolution, and transmission electron microscopy in combination with molecular biology techniques. Moreover, as EPS8 is remarkably specific to the distal tips of microvilli, this specificity can be harnessed to define other distal tip complex proteins with a biotin proximity labeling approach. By creating a chimeric fusion with the biotin ligase BioID2 (EPS8-BioID2), proteins residing in the distal tip complex can be biotinylated, isolated, and identified by mass spectrometry. As such, revealing the identity of distal tip complex proteins would provide insight into the general mechanism of microvillar growth, a process essential for normal brush border physiology. Thus, understanding mechanisms underlying microvillar growth can provide insight into diseases where microvillar morphology is compromised, such as microvillus inclusion disease, celiac disease, and Chron?s disease.